Nanopores can be used for biochemical analysis, mostly structural analysis of linear organic molecules. Whereas originally bio-nanopores were used, these are not stable and typically can only be used once. Consequently, solid-state nanopores which do not suffer from this problem are preferred for nanopore sequencing technology. Solid-state nanopores are holes fabricated artificially in a membrane with diameter in the range (0.1 nm-999 nm). Molecular sequencing in such nanopore relies amongst others on translocation of the target molecule through the nanopore. A particular application of nanopores that is often quoted is DNA sequencing. Transduction and recognition are performed sequentially and in real-time on segments of the molecule. Translocation is achieved passively or (with greater control) actively. Active translocation can be achieved by means of electrophoresis in which a voltage is applied on (two) electrodes placed in fluidic reservoirs separated by the membrane, the resulting electrical field then propels the charged molecule through the pore.
Various electric or electronic interactions can be exploited for sensing in the pore. DNA translocation events are routinely detected by measurement of the ion current through the nanopore. The presence of a DNA molecule in the pore leads to an increase or decrease of the ionic current. More particularly, different base molecules on the DNA strand result in a different blockage effect to the ion flux present at the nanopore. By recording these small changes in impedance, one can get information on the DNA sequence. Provided such measurements can be performed with sufficient sensitivity, information on structural or chemical composition of the molecule could be harvested from ionic current data. In another method, electrodes are mounted in the pore and electronic properties of the molecule are measured there. When a voltage is applied across the electrodes, an electronic current can flow stimulated by quantum mechanical electron tunneling via the electronic states of the molecule. Such mechanism provides chemical specificity. In yet another approach, capacitive modulations are sensed.
One challenge in nanopore sequencing technology is to lower the translocation speed of the DNA strand as the translocation speed currently is so fast that the signals from single base molecules on the DNA strand are not readable. A number of solutions have been provided in the prior art.
In “Slowing DNA translocation in a solid-state nanopore”, Fologea describes a technique for reducing the DNA translocation speeds by controlling the environmental conditions, more particularly by controlling the electrolyte temperature, salt concentration, viscosity and the electrical bias voltage across the nanopore. It is shown that adjusting these environmental parameters results in a significant result of the DNA translocation speed.
Another example for decelerating translocation inside a nanopore is chemical functionalisation of the nanopore, as e.g. described by Clake et al. in Nat. Nanotech. 2009 p 265. Chemical interaction between the functionalised nanopore and the DNA to be measured then results in deceleration.
Still another solution for slowing down DNA translocation is the use of an optical tweezer. In this indirect way for controlling DNA translocation, DNA fragments are bound to a bead which can be trapped at or close to the nanopore using the optical tweezer. A disadvantage of such a technique is the requirement for bounding the DNA fragment to the bead, which may influence the DNA, which requires additional equipment and beads, and which requires an additional processing step.